Extent of apoptosis in ovarian serous carcinoma: relation to mitotic and proliferative indices, p53 expression, and survival.

AIMS: To assess the extent of apoptosis in ovarian serous carcinoma and to examine possible relations between apoptosis, cell proliferation, p53 overexpression, and patient survival. METHODS: Apoptotic and mitotic indices were obtained by examining haematoxylin and eosin stained sections from 30 patients with ovarian serous carcinoma. Apoptosis was also evaluated semiquantitatively by in situ end labelling of fragmented DNA. Expression of p53 and determination of Ki-67 labelling indices were based on immunohistochemical staining. Clinical details were obtained from patients' clinical records. For statistical analysis, Fisher's exact test, parametric (Pearson) linear correlations, and the Kaplan-Meier method were used. RESULTS: The mean apoptotic index was 1.3% (range 0.02-3.9%), the mean mitotic index was 0.4% (range 0.02-1.1%), and the mean Ki-67 labelling index was 16% (range 4-32%). There were significant correlations between the apoptotic and mitotic indices (p < 0.0205) and between the mitotic and Ki-67 labelling indices (p < 0.024). There was a significant correlation between a high apoptotic index and poor prognosis (p < 0.02). p53 was overexpressed in 16 cases but the extent of apoptosis and outcome were both independent of p53 status. CONCLUSIONS: These results suggest that regulation of apoptosis is an integral component of tumour cell kinetics in ovarian serous carcinoma, and that increased apoptosis is indicative of aggressive tumour growth. p53 expression did not correlate with altered apoptosis, but the possibility of an attenuated apoptotic response to subsequent DNA damage by anticancer agents is not excluded.     

Expression profiling of colorectal carcinomas using tissue microarrays: cell cycle regulatory proteins p21, p27, and p53 as immunohistochemical prognostic markers in univariate and multivariate analysis.

With the rapidly growing understanding of tumor biology, molecular staging of cancer is expected to improve prognostication. This would be particularly important for cancers amenable to adjuvant treatment, such as colorectal carcinomas. To generate data for this, the tissue microarray technique may prove useful. Tissue microarrays were constructed with triplicate cores (0.6 mm diameter) from the invasive margins of a consecutive single-institution series of 184 colorectal carcinomas. Immunostaining for p53, p21, p27, Ecadherin, and beta-catenin was scored. Tumor cell proliferation was assessed by mitotic indices and Ki-67 labeling, apoptosis by quantification of apoptotic bodies. Reduced nuclear immunostaining for p21 (<10%) and p27 (< or =50%) and reduced membranous expression of Ecadherin were significantly associated with a poorer clinical course by univariate analysis. beta-catenin immunostaining had no prognostic impact. Mitotic and apoptotic indices as well as Ki-67 labeling below the median were indicators of poor prognosis. Complete absence of p53 nuclear staining was a significant adverse prognostic factor. By Cox regression, p53 = 0%, p53 = 0%, in combination with p27 < or = 50%, the mitotic index and the combined mitotic and apoptotic index added prognostic information to UICC stage. The authors found that growth pattern, lymphohistiocytic response, lymphatic permeation, and venous spread, too, each was a strong prognosticator in addition to UICC stage. The results support that tissue microarrays are a useful tool for screening immunohistochemical markers for prognostic use. An immunopanel of p21, p27, and p53 could be useful for prognostication in colorectal carcinoma in addition to UICC stage.     

Neoadjuvant chemotherapy in cervical carcinoma: regulators of cell cycle, apoptosis, and proliferation as determinants of response to therapy and disease outcome.

To evaluate whether cellular markers predict the responsiveness to neoadjuvant chemotherapy (NAC) in cervical cancer, 21 patients with stages I and II cervical carcinomas treated by NAC before surgery were followed up for a mean of 52.3 months. Pre-NAC biopsy and operative specimens were subjected to counting of apoptotic (AI/V) and mitotic (MI/V) indices, detection of human papillomavirus (HPV) DNA, and immunohistochemical analysis of cell cycle and proliferation markers (p21, p53, pRb, proliferating cell nuclear antigen [PCNA], Ki-67) and multidrug resistance gene (MDR1), as related to NAC response (RAC), recurrence-free (RFS), and overall (OS) survival. Adenosquamous histology and lymph node involvement were significant determinants of nonsurvival. All carcinomas contained HPV DNA. In univariate analysis, p21, pRb, and MDRI in the biopsy specimen and PCNA, Ki-67, and pRb in the surgical sample significantly predicted RAC, while age, AI/V number of lymph nodes removed, and MI/V predicted RFS. Highly significant predictors of OS were AI/V number of lymph nodes removed, post-NAC MDR1 expression, MI/V and recurrence. Multivariate analysis confirmed the strong post-NAC effects of histologic type, AI/V, and MDR1 expression for RFS, and recurrence, age, and Ki-67 expression for OS. NAC responders with slightly decreased AI/V and increased MI/V had a poor prognosis.     

卵巢低、高级别浆液性癌的临床病理分析及Pax2、p53和Ki-67表达的意义

目的 初步评估卵巢浆液性癌的两级分级系统在病理诊断和预后判断中的应用价值,探讨低、高级别浆液性癌Pax2、p53和Ki-67的表达及其与预后的相关性.方法 依照两级分级系统对卵巢浆液性癌进行分级,分别选取低级别癌38例,高级别癌100例,观察其临床病理特点,并采用免疫组织化学EnVision法检测癌组织中Pax2、p53和Ki-67的表达情况.结果 (1)低级别组的总生存时间、无病生存时间和5年生存率均显著优于高级别组(P＜0.05).(2)低级别组Pax2的阳性率显著高于高级别组(65.8%比13.0%,P＜0.05),而p53强阳性率显著低于高级别组(13.2%比55.0%,P＜0.05),Ki-67阳性指数显著低于高级别组(13.7%比42.1%,P＜0.05).(3)Pax2阳性组的总生存时间和5年生存率均显著优于阴性组(P＜0.05),而无病生存时间的差异无统计学意义(P＞0.05).p53和Ki-67阳性指数与生存时间的差异无统计学意义(P＞0.05).结论 两级分级系统对卵巢浆液性癌的预后判断有提示意义,在临床病理诊断中具有可行性.Pax2、p53和Ki-67在卵巢低、高级别浆液性癌中的表达差异有统计学意义.与p53和Ki-67相比,Pax2更有可能成为卵巢浆液性癌的一个预后相关指标.

Abstract：

Objective To evaluate the two-tier system for the grading of ovarian serous carcinomas,and to analyze Pax2, p53, Ki-67 protein expression and their prognostic values for low- and high-grade ovarian serous carcinomas. Methods A total of 38 cases of low-grade and 100 cases of high-grade ovarian serous carcinomas were selected based on the two-tier grading system. Innnunohistochemistry was used to detect Pax2, p53 and Ki-67 protein expression in all cases. Correlation of the two-tier system with immunohistochemical results and prognostic parameters were performed. Results ( 1 ) The overall survival,disease-free survival and 5-year survival rates were significantly higher in the low-grade serous carcinoma cases than in the high-grade cases ( P ＜ 0. 05 ). (2) Significant differences in protein expressions were found between the low- and high-grade serous carcinomas. The high-grade serous carcinomas had a significantly higher expression level of p53 (55.0% vs 13. 2%, P ＜ 0. 05 ) and Ki-67 (42. 1% vs 13.7%, P ＜ 0. 05 ),while low-grade carcinomas had a significantly higher expression level of Pax2 (65. 8% vs 13.0%,P ＜ 0. 05 ). (3) Pax2 positive cases had a significantly better overall survival and 5-year survival rates than Pax2 negative cases ( P ＜ 0. 05 ). The expressions of p53 and Ki-67 were found to have little correlation with overall survival and disease-free survival( P ＞ 0. 05 ). Conclusions The two-tier system for the grading of ovarian serous carcinomas has a good prognostic value. There are significantly differences in expressions of Pax2, p53 and Ki-67 between low- and high-grade ovarian serous carcinomas. Compared with p53 and Ki-67, Pax2 is likely a better prognostic indicator for ovarian serous carcinoma.     

Prognostic significance of the mitotic index using the mitosis marker anti-phosphohistone H3 in meningiomas

Mitotic activity is one of the most reliable prognostic factors in meningiomas. The identification of mitotic figures (MFs) and the areas of highest mitotic activity in H&E-stained slides is a tedious and subjective task. Therefore, we compared the results from immunostaining for the mitosis-specific antibody anti-phosphohistone H3 (PHH3 mitotic index [MI]) with standard MF counts (H&E MI) and the Ki-67 labeling index (LI). The relationship between these proliferation indices and prognosis was investigated in a retrospective series of 265 meningiomas. The PHH3 staining method yielded greater sensitivity in the detection of MFs and facilitated MF counting. Mitotic thresholds of H&E MI of 4 or more per 10 high-power fields (HPF) and PHH3 MI of 6 or more per 10 HPF were found as the most appropriate prognostic cutoff values for the prediction of recurrence-free survival. All 3 proliferation indices were univariately associated with recurrences and deaths. In contrast with the Ki-67 LI, H&E MI and PHH3 MI also remained as independent predictors in the multivariate Cox hazards modeling (P = .0007 and P = .0004, respectively).     

Apoptosis occurs independently of bcl-2 and p53 over-expression in non-small cell lung carcinoma

Certain oncogenes and tumour suppressor genes are known to modulate apoptosis. To investigate whether over-expressed bcl-2 and abnormally stabilized p53 are associated with reduced apoptosis in paraffin sections of non-small cell lung carcinoma, apoptotic, mitotic, and Ki-67 labelling indices were determined and correlated with bcl-2 and p53 immunoreactivity in 54 squamous cell carcinomas and 22 adenocarcinomas. Nineteen squamous cell carcinomas (35.2%) showed over-expression of bcl-2, but all 22 adenocarcinomas were bcl-2 negative. Thirty-seven squamous cell carcinomas (68.5%) and 13 adenocarcinomas (59.1%) showed p53 over-expression. Apoptotic tumour cells were identified among p53 positive and bcl-2 positive tumour cells. There was a significant linear correlation between apoptotic indices and mitotic indices. bcl-2 over-expression and p53 over-expression were not associated with attenuated apoptosis, or altered mitotic or Ki-67 labelling indices in either tumour type. Neither bcl-2 nor p53 was of prognostic significance. These results suggest that apoptosis in non-small cell lung carcinoma occurs independently, and is not modulated primarily by, bcl-2 or p53. It is likely that the effects on apoptosis of bcl-2 and p53 are countered by those of other oncogene products and/or additional factors that regulate apoptosis in vivo     

Low levels of apoptosis and proliferative activity in colorectal villous tumors: Comparison with tubular tumors

In order to clarlfy the cell kinetics of colorectal villous tumors (VT), 21 villous adenomatous areas and 12 carcinomatous areas within villous adenomas were investigated for proliferative activity and apoptosis and compared with a series of 41 tubular tumors (TT), demonstrating elements of intramucosal carcinomas as well as tubular adenomas (so-called carcinoma in tubular adenoma). Proliferation was estimated in terms of KI-67 labeling indices and mitotic indices, and apoptosis was assessed by DNA nick-end labeling to give apoptotic Indices. Apoptotic indices of villous adenomatous and carcinomatous regions were significantly lower than the values for their tubular counterparts. Kl-67 labeling indices were also significantly lower for adenoma components. Apoptotic indices, Ki-67 labeling indices and mitotic Indices increased with atypia raised in tubular adenoma components. Correlations of mitotic indices with apoptotic indices, Ki-67 labeling Indices with apoptotic indices and mitotic Indices with Ki-67 labeling indices were found for each villous tumor group and tubular tumor group, and the apoptosis and proliferation ratios for villous tumors were relatively low, suggesting a tendency for greater growth due to less cell deletion. Although this is only one of the biological features of villous tumor groups, it might play a major role in generation of malignancy.     

Immunohistochemical markers of cell cycle control applied to ovarian and primary peritoneal surface epithelial neoplasms: p21(WAF1/CIP1) predicts survival and good response to platinin-based chemotherapy.

Immunohistochemistry for p53, p21(WAF1/CIP1), and Ki-67 provides insight into the molecular events controlling the cell cycle. We tested the hypothesis that these cell cycle markers will aid in the clinical evaluation of ovarian and primary peritoneal surface epithelial neoplasms (SENs). Paraffin sections from a retrospective surgical series of 117 SENs were immunostained with anti-p53 (clone DO7, Novacastra Laboratories, UK), anti-p21(WAF1/CIP1) (clone EA10, Oncogene Science, Cambridge, MA), and anti-Ki-67 (clone MIB-1, Immunotech, Westbrook, ME). The Ki-67 proliferation index (Ki-67PI) and immunoreactivity were evaluated. One hundred seventeen SENs reacted as follows: p53 50%+ and p21(WAF1/CIP1) 65%+. Ki-67PI ranged from 4% to 88% (mean/median = 44/46%). p53 reactivity associated with transitional cell histology, decreased p21(WAF1/CIP1) staining, increased Ki-67PI, architectural/nuclear grade, and stage (P < .05, 1 x 10(-7), .01, .05/.0001, .001,). p21(WAF1/CIP1) staining was associated with endometrioid/clear cell histology, decreased Ki-67PI, architectural/nuclear grade, and stage (P < 05/.05, .05, .01/1 x 10(-8), 1 x 10(-5)). Ki-67PI associated with increased architectural/nuclear grade but not mucinous histology (P < 1 x 10(-5)/1 x 10(-6), .01). Sixty-seven patients had disease at last follow-up; 53 were dead of disease at 0 to 67 months (mean/median, 21/18), and 14 were alive with disease at 12 to 224 months (mean/median, 56/40). Fifty patients were disease free at 5 to 214 months (mean/median, 59/41). Predictors of survival include decreased Ki-67PI, stage, architectural/nuclear grade (P < 1 x 10(-6), 1 x 10(-10), 1 x 10(-10)/.005) and p21(WAF1/CIP1) IMS (multivariate P < 1 x 10(-6)). p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases necessary for cell cycle progression, functions as a key checkpoint in cell cycle control. Immunoreactivity for p21(WAF1/CIP1) provides prognostic information independent of other histological and clinical predictors, p53 IMS, and Ki-67PI in this series of 117 PTs with SENs. Our preliminary data suggest an interrelationship between p21(WAF1/CIP1) expression and an effective clinical response to platinin-based chemotherapy, both associated with apoptosis. Further investigation seems warranted.     

Immunohistochemical detection of dUTPase in intracranial tumors

The nuclear isoform of deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, OMIM *601266, EC 3.6.1.23) is immunohistochemically detectable in all proliferating tissues and may thus be a useful adjunct for the grading of tumors analogous to Ki-67 labeling. A hundred and twenty-seven human intracranial tumors, including 56 astrocytomas, 12 oligodendrogliomas, 8 oligoastrocytomas, 34 meningiomas, 7 ependymomas, and 10 metastatic carcinomas, were stained using the monoclonal rat anti-human dUTPase antibody (clone 3E6) with formalin-fixed and paraffin-embedded tissue. The labeling indices were compared with those obtained with the proliferation marker Ki-67 on parallel tissue sections. All tumors contained dUTPase-positive nuclei, whereas the percentage of positive tumor cells generally increased with grade of malignancy. Meningiomas of higher grades, i.e., World Health Organization (WHO) grades II and III, contained additional cells with cytoplasmic reactivity. There were usually fewer dUTPase- than Ki-67-positive nuclei detectable. Unlike Ki-67, dUTPase was not detectable in mitotic figures. Labeling indices for dUTPase, but not for Ki-67, showed significant differences between all 3 WHO grades of diffuse astrocytomas. In summary, dUTPase staining provides a useful measure of cell proliferation distinct from that offered by Ki-67 labeling. It proved particularly useful for the evaluation of diffuse astrocytomas.     

Loss of DCC expression in astrocytomas: relation to p53 abnormalities, cell kinetics, and survival

AIMS: Although frequent reduction or loss of DCC (deleted in colorectal carcinomas) has been demonstrated in gliomas, the association with cell kinetics and survival is still unclear. METHODS: A total of 119 astrocytomas, comprising 39 grade IV, 36 grade III, and 44 low grade tumours, were immunohistochemically investigated, along with 26 normal adult brain samples and two fetal brains. The results were compared with p53 abnormalities, Ki-67 labelling index (LI), mitotic index (MI), apoptotic index (AI), and survival. RESULTS: In normal adult and fetal brain tissues, DCC expression was detected in mature and terminally differentiated neuronal cells but not glial elements. In astrocytomas, whereas DCC expression was still clearly shown with low grade malignancy, DCC scores were significantly decreased in high histological grade malignancy, along with an increase in cell kinetics determined by AI, MI, and Ki-67 LI values. In addition, p53 LI values were significantly increased, although a direct link between DCC scores and p53 LI values was not evident. Univariate analysis revealed that high DCC scores and low p53 LI values were closely related to a favourable outcome for astrocytoma, although only the AI was an independent prognostic factor. CONCLUSIONS: The loss of DCC expression may be closely related to changes in cell kinetics and tumour phenotype in astrocytomas, independent of p53 abnormalities.     

Proliferative activity as revealed by Ki-67 in uterine adenocarcinoma of endometrioid type: Comparison of tumours from patients with and without previous oestrogen therapy

The Ki-67 antibody recognizes a nuclear antigen related to cell proliferation, which in some studies has been shown to reflect the aggressiveness of tumours. The percentage of Ki-67-positive cells was estimated by immunohistochemistry on frozen tissue sections from 73 adenocarcinomas of endometrioid type (EC) (40 tumours from patients who had never received postmenopausai oestrogen treatment and 33 tumours from patients with previous postmenopausal oestrogen treatment). The Ki-67 content was weakly but significantly (P<0.05) correlated to nuclear grade, architectural grade, and crude mitotic count, but not to stage or progesterone receptors. Ki-67 expression in EC from patients with previous oestrogen therapy was much lower (median 10 per cent Ki-67) than that in EC from patients who had never received oestrogen treatment (median 24 per cent Ki-67), suggesting that a prognostic cut-off point may be different in tumours from the two groups of patients. The mitotic count discriminated the two groups of patients to a much smaller degree. It is also shown that a quick qualitative Ki-67 estimate can replace the time-consuming quantitative assessment.     

Immunohistochemical analysis of Ki-67, p53, p21, and p27 in benign and malignant apocrine lesions of the breast: its correlation to histologic findings in 43 cases.

We examined Ki-67, p53, p21, and p27 immunolocalization in 43 cases of apocrine lesions of the breast and correlated these findings with histologic parameters to understand their biologic significance. Twenty cases were benign, 1 case was borderline, and 22 cases were diagnosed as malignant, including 9 intraductal and 13 invasive apocrine carcinomas. Both the ratio of Ki-67-positive cases (17 of 21 [88.9%] versus 1 of 19 [5.3%]; P < .001) and the Ki-67 labeling index of positive cases examined (15.0% versus 2.7%; P < .005) were significantly higher in malignant than in benign apocrine lesions. None of the benign or borderline cases was immunohistochemically positive for p53, but 15 of 22 malignant cases (68.2%) demonstrated p53 (P < .001). In addition, the ratio of p53-positive cases was significantly higher in high nuclear grade cases (11 of 13 [84.6%]) than in intermediate nuclear grade cases (4 of 9 [44.4%]; P < .05). P53 immunoreactivity was also positively correlated with the nuclear grade of carcinoma cases examined in this study. Neither p21 nor p27 demonstrated any correlation with histologic parameters or findings of the apocrine lesions. Results of these studies suggest that Ki-67 and p53 may be good markers for differentiation between benign and malignant breast apocrine lesions.     

The proliferation markers Ki-67/MIB-1, phosphohistone H3, and survivin may contribute in the identification of aggressive ovarian carcinomas

The identification of new proliferation markers could have clinical implications in ovarian carcinoma by stratifying patients for treatment and follow-up. The aim of this study was to evaluate the diagnostic and prognostic value of the proliferation markers Ki-67/MIB-1, phosphorylated histone H3 (PHH3), and survivin in epithelial ovarian tumors. Ninety women with a pelvic mass who underwent surgery at the Department of Gynecological Oncology were included: 68 ovarian carcinomas, 11 borderline tumors, and 11 ovarian cystadenomas. We performed mitotic count and immunohistochemical analyses of Ki-67/MIB-1, PHH3, and survivin, related to clinicopathological parameters. Uni- and multivariate analyses of five-year overall survival were performed. We found statistically significant correlations between mitotic count, Ki-67/MIB-1, PHH3, and survivin. The expression of all proliferation markers was significantly higher in the carcinomas than in the borderline and benign tumors (p<0.05). There was, however an overlap of indices between the different malignancy groups. Women with advanced stage cancers (FIGO stage III and IV) had significantly higher tumor expression of all markers compared to patients with early stage cancers (FIGO stage I and II). Women with advanced disease and complete chemotherapy response had higher Ki67/MIB-1 expression than women without complete chemotherapy response. All markers had an impact on survival in the univariate analyses. In the multivariate analysis, however, only age and stage of disease reached statistical significance as prognostic factors. In conclusion, the proliferation markers Ki-67/MIB-1, PHH3, and survivin are positively correlated with each other and with tumor grade, and may contribute in the identification of aggressive ovarian carcinomas.     

Impact of p53 and Ki-67 in predicting recurrence and progression of superficial (pTa and pT1) urothelial cell carcinomas of urinary bladder

In predicting the aggressive behavior of bladder tumors, the histopathological characteristics of grade and invasive stage are of principal importance. However, for predicting tumor recurrence and progression, these are sufficient only to a limited extent, particularly in the case of superficial (pTa and pT1) urothelial cell carcinomas. New prognostic factors are therefore needed to avoid either insufficient or excessive treatment. In this retrospective study, we investigated the prognostic value of the p53 and Ki-67 immunoreactivity indices. The present study included 118 superficial urinary bladder tumors consisting of 58 recurrent and 60 non-recurrent cases. Twenty of the recurrent tumors progressed into a higher grade and/or invasive stage. Paraffin immunohistochemical analysis was carried out using anti-p53 and anti-Ki-67 antibodies on the initial tumor tissues. We concluded that there is a highly significant relationship between the p53 and Ki-67 immunoreactivities and the histological grade and pathological stage of the tumors (P < 0.0001). We observed a significant relationship between the presence of recurrence and progression and the p53 immunoreactivity index (P < 0.01 and P = 0.017, respectively) and Ki-67 immunoreactivity index (P < 0.0001 and P = 0.046, respectively). Positivity for p53 and Ki-67 can demonstrate the risk of recurrence (p53: sensitivity = 76%, specificity = 58%; Ki-67: sensitivity = 86%, specificity = 48%) and progression (p53: sensitivity = 80%, specificity = 46%; Ki-67: sensitivity = 85%, specificity = 36%; ). We believe that both of these immunohistochemical markers can be considered valuable in addition to classical histopathological prognostic parameters for predicting recurrence and progression risks.     

Are polyploid giant cancer cells in high grade serous carcinoma of the ovary blastomere-like cancer stem cells?

Polyploid giant cancer cells, either multinucleated or mononucleated, in high grade serous carcinoma of the ovary have been previously recognized. Different theories including degenerative changes or an important step in the development of high grade serous carcinoma have been proposed. Here we investigate possible explanations for the presence of polyploid giant cancer cells in high grade serous carcinoma. We reviewed 33 cases of ovarian high grade serous carcinoma (12 stage I, 7 stage II, and 14 stage III). We counted the number of polyploid giant cancer cells in 20 consecutive 10× fields. In 11 cases where polyploid giant cancer cells were easily found, immunohistochemistry for Ki67, p53, and OCT 3/4 was performed. Patients with polyploid giant cancer cells were older than those without. Polyploid giant cancer cells were more frequent in stage I lesions (75%) than in stages II or III (57% in both) and less frequent in metastases compared with primary ovarian tumors. Mitotic figures were present in regular sized cells but were absent in polyploid giant cancer cells. OCT3/4 was negative in all cases assessed. In 8 cases, more than 70% of the mononuclear cells were positive for Ki-67, similar to the percentage of Ki-67 positive cells in polyploid giant cancer cells. p53 had a perfect correlation in regular sized cancer cells and in polyploid giant cancer cells.Polyploid giant cancer cells are neither degenerative cells nor traditional cancer stem cells but most probably represent an intermediate step between stem cells and mature tumor cells formed by endoreplication.     

Enhanced expression of Ki-67, topoisomerase IIα, PCNA, p53 and p21 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi

To investigate the effect of ultraviolet (UV) irradiation on the expression of cell cycle-associated proteins, melanocytic nevi from healthy volunteers were partially covered, irradiated with a defined UV dose, and excised 1 week thereafter. The irradiated and the protected parts were examined separately by conventional microscopy and immunohistochemistry using the antibodies Ki-S11 (Ki-67), Ki-S7 (topoisomerase IIα), PC10 (proliferating cell nuclear antigen [PCna]), DO-7 (p53), 6B6 (p21^WAF1/Cip1), and the melanocytic marker HMB-45. DNA nick-end labeling was used as a marker of apoptosis. Irradiation resulted in morphological changes and increased HMB-45 reactivity. Proliferation, as assessed by Ki-67 and topoisomerase IIα expression, was also clearly enhanced in the UV-exposed areas. This was confirmed by the appearance of occasional mitotic figures. PCNA expression levels markedly exceeded those of the proliferation markers and did not correlate with the latter in most cases. p21 immunolabeling indices were also consistently augmented after UV exposure; hence it is likely that growth-inhibitory mechanisms partly compensate for the proliferative impulse, and the disproportional rise in PCNA expression probably reflects DNA repair activity. Enhanced p53 immunostaining in four cases suggests that the induction of p21 after irradiation may be p53 mediated, whereas no concomitant apoptotic events were observed. We conclude that UV light can stimulate the proliferative activity of melanocytes in melanocytic nevi, but that simultaneously cell cycle inhibitors are activated to permit DNA repair.     

Proliferation indices in spontaneous canine lung cancer: proliferating cell nuclear antigen (PCNA), Ki-67 (MIB1) and mitotic counts

Proliferation indices were measured for specimens from 55 spontaneous canine lung tumours, collected by surgical biopsy from clinical patients and archived in paraffin wax blocks. These indices were then related to the mitotic index and histological type of the tumour. Proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB1) proteins were detected immunohistochemically with a biotin-streptavidin amplified detection system on a representative tissue section from each tumour. Five adjacent, non-overlapping fields were selected at random, and 200 cells per field were examined in each section. For PCNA, cells were classified subjectively into negative, weak or strong reactivity groups, based on nuclear staining. MIB1 cells were classified as negative or positive, based on nuclear staining. Mitotic figures were counted in anti-PCNA-labelled and anti-MIB1-labelled sections, in the same tumour areas as those in which indices were established. Mitotic counts were also done on haematoxylin and eosin-stained tissue sections. Linear regression analysis showed that all PCNA, MIB1 and mitotic indices had highly significant positive correlations (P<0.0001) with each other. Adenosquamous and squamous cell carcinomas differed from other histological tumour types in having significantly higher proliferation indices. These data suggest that growth rates for lung tumours in the dog vary according to histological type. On the basis of differences in proliferation indices and the distribution of immunoreactivity between histological subtypes in this study, it would seem that immunohistochemical detection of PCNA and MIB/1 reactivity and analysis of mitotic figures in routinely processed tissues may be useful in the diagnosis of lung tumours. 1999 W.B. Saunders and Company Ltd.     

Enhanced epithelial cell turnover associated with p53 accumulation and high p21WAF1/CIP1 expression in ulcerative colitis.

To cast light on accelerated epithelial cell turnover as an important risk factor of dysplasia and carcinoma development in patients with long-standing ulcerative colitis (UC), we examined cell proliferation and cell death, as well as expression of apoptosis-related markers, including p53 and p21WAF1/CIP1, in a series of cases. Biopsy specimens (n = 176; 84, active phase; 92, remission) were endoscopically obtained from 25 Japanese patients with UC. As controls, 68 biopsy specimens of normal mucosa were also examined from 27 Japanese patients with colon polyps. We counted the numbers of mitoses, apoptotic bodies, Ki-67-immunoreactive cells, and p21WAF1/CIP1-immunoreactive cells per 1000 crypt cells and the numbers of p53-positive cells per crypt. All of the indices in active UC were significantly higher than in either remitting UC cases or normal cases (mean mitotic index = 0.52, 0.28, and 0.15%, respectively; apoptotic index = 5.40, 2.91, and 1.30%, respectively; Ki-67 labeling index = 39.5, 28.3, and 26.8%, respectively; p21WAF1/CIP1 labeling index = 33.6, 20.0, and 19.0%, respectively; p53 labeling index = 0.66, 0.13, 0.13 per crypt, respectively). In addition, the mitotic, apoptotic, and Ki-67 labeling indices were increased in remitting UC of more than 10 years' duration, in comparison with those of less than 10 years' duration or the normal group. Immunostaining of serial sections revealed a small number of crypt cells coexpressing p53 and p21WAF1/CIP1. Increases in both epithelial cell proliferation and cell death, partially associated with p53 accumulation and high p21WAF1/CIP1 expression, are thus characteristic of active phase UC, as well as in remission of long-standing UC. Accelerated epithelial cell turnover caused by chronic inflammation and epithelial damage might predispose the mucosa to DNA damage, resulting in an elevated risk of mutation in line with dysplasia and carcinoma development in patients with UC.     

Grading system for lymph vessel tumor emboli for prediction of the outcome of invasive ductal carcinoma of the breast

There are no suitable histologic diagnostic clues for determining the true biological malignancy of invasive ductal carcinomas associated with lymph vessel tumor emboli. The purpose of this study was to devise a grading system for lymph vessel tumor emboli in invasive ductal carcinomas that would allow accurate prediction of the outcome of invasive ductal carcinoma patients with lymph vessel invasion. We classified 393 invasive ductal carcinomas into the following 4 grades according to the number of mitotic and apoptotic figures in tumor cells in lymph vessels at 1 high-power field: grade 0, no lymph vessel invasion; grade 1, absence of mitotic and apoptotic figures, presence of any number of mitotic figures and absence of apoptotic figures, or absence of mitotic figures and presence of any number of apoptotic figures; grade 2, 1 to 4 mitotic figures and 1 or more of apoptotic figures, or 1 or more of mitotic figures and 1 to 6 apoptotic figures; and grade 3, more than 4 mitotic figures and more than 6 apoptotic figures. The mortality rate increased with the grade, and the mortality rate of patients with grade 3 lymph vessel tumor emboli was more than 70%. Multivariate analyses with well-known prognostic factors demonstrated that grade 3 lymph vessel tumor emboli significantly increased the hazard rates for tumor recurrence, and tumor death independent of adjuvant therapy status, nodal status, or invasive tumor size. The grading system for lymph vessel tumor emboli is the best histologic grading system for accurately predicting the outcome of patients with invasive ductal carcinoma of the breast.     

Assessment of proliferative activity in ovarian neoplasms by flow and static cytometry. Correlation with prognostic features.

We undertook a prospective flow and static cytometric study of proliferative activity in 74 malignant, borderline and benign ovarian neoplasms. Proliferative activity as assessed by S phase fraction (SPF), and immunostaining of tissue sections with Ki-67 antibody was compared with prognostically important clinicopathologic features. Malignant neoplasms had higher median percentage Ki-67 staining (27.6%) than borderline (12.3%) and benign (2.7%) tumors (P less than 0.05). Percentage Ki-67 staining correlated with SPF, DNA index, architectural grade, nucleolar grade, and mitotic count in malignant tumors; with nucleolar grade in benign tumors and with none of these variables in borderline tumors. Similarly, malignant neoplasms had a higher median SPF (11.5%) than borderline (3.4%) and benign (2.9%) neoplasms (P less than 0.05). In malignant neoplasms, SPF correlated with percentage Ki-67 staining, DNA index, age, and stage, but with none of these in borderline or benign neoplasms.